1. Field of the Invention
The present invention relates to a method of purifying an antibody employing protein affinity chromatography. More specifically, the present invention relates to an elution buffer component useful in the method.
2. Brief Description of the Related Art
Antibodies are extremely useful in drug formulations, as clinical examination reagents, and as research reagents. Hence, demand for them is increasing. Staphylococcal Protein A (“protein A” hereinafter), an Fc receptor derived from a microbe, exhibits extremely high affinity for multiple antibody Fc domains. Thus, affinity chromatography employing immobilized protein A as a ligand on a support has become the core technique in industrial-scale antibody manufacturing (for example, see The Production of Monoclonal Antibodies. In Birch, J. R. and Lennox, E. S. (ed.); Monoclonal Antibodies, Principles and Applications, p. 231-265, London: Wiley Liss, Inc., 1995). The high affinity of protein A increases production efficiency per unit time, and contributes to the elimination of impurities which originate in the starting materials to a high degree. However, problems due to antibody characteristics remain in Protein A affinity chromatography, limiting antibody production. The present invention solves these problems relating to antibody characteristics and affords improvement toward more stable manufacturing of antibody.
Protein A exhibits extremely high affinity for Fc domains at neutral pH. Thus, starting material containing antibody to be purified can be loaded onto a column packed with support upon which protein A has been immobilized at neutral pH. After thorough washing with neutral pH buffer solution to remove impurities in the starting materials, the antibody can be desorbed from the column with an elution buffer solution of acidic pH, specifically, from pH 2.5 to less than pH 4. Impurities deriving from the starting materials that remain in the antibody after desorbtion at acidic pH are present on the order of only several hundred ppm, and the various immobilized protein A supports that can be used for industrial production are known to present no difference in basic performance (for example, see R. L. Fahrmer, D. H. Whitney, M. Vandertaan, G. Blank, Biotech. Appl. Biochem (1999) 30, 121-128 and R. Hahn, R. Schlegel, A. Jungbauer, J. Chromatogr. B., (2003) 790, 35-51). Currently, immobilized protein A supports suitable for use in industrial antibody production are commercially available from Amersham Biosciences, Inc., Millipore, PE Biosystems Corp., and the like. However, common problems are encountered when purifying antibody with these supports. The antibody that is desorbed from the support and recovered comes in contact with a highly acidic buffer solution, altering the tertiary structure of the antibody.
Furthermore, association and aggregation tend to occur during frequently. Extensive research into changes in antibody structure caused by acidic pH has been conducted. However, resolution of the issues regarding structural change and the association and aggregation reactions has yet to be proposed. The fact that exposure to acidic pH causes certain problems in antibodies has been reported chiefly as a practical problem. See J. M. Sarciax et al., Journal of Pharmaceutical Sciences, 88 (1999), 1354-1361; and M. Paborji et al., Pharmaceutical Research, 11 (1994), 764-771. The fact that acidic pH causes changes in the tertiary structure of an antibody has been demonstrated in a number of experiments. See Buchner et al., Biochemistry, 30 (1991), 6922-6929; Buchner et al., Biophysical Journal 78 (2000), 394-404; and Buchner et al., Biochimica et Biophysica Acta, 1431(1999), 120-131. Vlasov et al. reports the effect that even when an antibody is titrated to neutral pH without having undergone association or aggregation as a result of contact with acidic pH, the original antibody structure cannot be restored. See Vlasov et al., Biochemistry (Moscow), 61 (1996), 155-171; Vlasov et al., Immunology Letters, 43 (1994), 149-152; Vlasov et al., FEBS Letters, 361 (1995), 173-175. Since the goal of purification is to retain the quality of antibody, acidic pH exposure is extremely undesirable. Therefore, there exists the need for a new refinement technique that does not require exposure to acidic pH.
Extensive research has been conducted into solving the above-stated problem. A number of examples of such research will be summarized and the problems of prior art will be enumerated below.
(1) Methods of recovering antibody from protein A in neutral buffer solution Based on the knowledge that tyrosine residues participate in the binding of protein A and antibody Fc domains, it was discovered that a dipeptide containing tyrosine (0.1 M glycyltyrosine, pH 7.0) could be used instead of an acidic elution buffer solution to desorb and recover antibody from protein A-binding support. See Bywater et al., Chromatogr. Synth. Biol. Polym., [Lect. Chem. Soc. Int. Symp.] 1978; Bywater et al., 1976 Meeting Proceedings, Volume 2, 337-340. However, there is a limit in that only about 20 to 35 percent of the bound antibody can be recovered under these conditions, rendering this method impractical.
Based on the knowledge that histidine residues participate in the binding of protein A and antibody Fc domains, it was discovered that not histidine alone but an imidazole solution (1 to 5 M, pH 6 to 9) corresponding to histidine residues could be employed instead of an acidic elution buffer solution to desorb and recover antibody from the protein A-binding support. See WO94/07912. However, this technique requires 1 to 5 M (3 M or better for good recovery) high-concentration imidazole elution. An imidazole solution is used as an elution buffer solution in metal chelate affinity chromatography for refinement of other substances such as fused proteins with a bound histidine tag are employed. However, it is well known by researchers in this field that imidazoles themselves denature proteins, and are unsuitable as elution buffer solutions for antibody purification. Thus, this method has also been found impractical.
(2) Methods of Employing Artificial Ligands in which the Amino Acid Sequence of Protein A has been Altered
S. Hober et al. focused on domain B as the domain at which protein A binds to antibody Fc, creating an artificial Z domain by changing a portion of the amino acid sequence of domain B. It has been shown that supports having immobilized domain Z as ligand binds well to antibody at neutral pH and permits efficient recovery of bound antibody using a mildly acidic buffer solution of pH 4.5 instead of an acidic elution buffer solution. See Hober et al., J. Chromatogr. 76 (2000) 233-244. However, the affinity of domain Z for antibody at neutral pH is much lower than that of protein A, and the decrease in production efficiency which accompanys the decrease in antibody binding capacity is a major problem. Further, the decrease in affinity relates to a decrease in the capability to eliminate impurities from the starting materials, so the problem of maintaining the quality of the product remains. Thus, this method has also been found to be impractical.
Based on X-ray crystal structure analysis of the protein A-antibody Fc domains complex, a number of variant forms of protein A were created in which the hydrophobic residues on the protein A molecule that participate in binding of the two molecules were replaced with histidine. Of these, whereby the Protein A leucine at residue numbers 21 and 79 was replaced with histidine, it was discovered that a decrease of pH 8 to only pH 5 in the buffer solution reduced affinity to antibody Fc domains to 1/50th the previous level. When the mildly acidic pH 5.0 buffer solution was employed instead of the acidic elution buffer solution, the antibody bound to the variant protein A could be effectively recovered. See M. G. Gore et al., Molecular Biotechnology 10 (1998), 9-16. However, the affinity of the variant protein A for antibody Fc domains at pH 8 is only ⅕th that of protein A. Similar to the above Z domain, there are significant problems such as decreased production efficiency and a decreased capability to eliminate impurities. Thus, this method has also been found impractical.
(3) Methods Employing Ligands Synthesized by Organic Chemistry Instead of Protein A
A compound was designed which undergoes a change in a hydrophobic property with a change in pH, irrespective of protein A. It was further discovered that this compound exhibits affinity for antibody Fc domains in a pH-dependent manner, and an antibody purification support was developed upon which this compound was bound as ligand. See Boschettir, Trends Biotechnol. 20 (2002), 333-337. This support can be purchased from Biosepra Corp. as MEP Hypercel. This support permits the recovery of bound antibody using a mildly acidic buffer solution (pH 4 to 5). However, the pH-dependent change in affinity is not as sharp as that of protein A, and desorption and recovery from the support when in concentrated form are impossible. Binding specificity is also much poorer than that of protein A, preventing the removal of impurities derived from the starting materials to a high degree. Although it is possible to set special washing conditions (using an organic acid or the like) suited to starting material purification to enhance the purification effect, the effect is still quite inferior to that of purification by protein A. Thus, this compound has not replaced protein A in industrial production.
A peptide mimicking the binding portion of protein A on antibody Fc domains was devised, and this structure was further developed, creating protein A-mimicking ligands completely synthesized by organic chemistry. See L. R. Down et al.; Mabsorbent®: Nature Biotechnology 16 (1998), 190-195. Antibody bound by these synthetic ligands can normally be desorbed and recovered at pH 3.0 with 10 mM sodium citrate, with the use of a neutral pH buffer solution being possible in the presence of ethylene glycol. However, since binding specificity is significantly poorer than that of protein A, the capability to eliminate impurities derived from starting materials is quite inferior to that of protein A. In a manner similar to that of the above MEP Hypercel, and since these synthetic ligands were primarily developed for use without protein A, a protein ligand, a decrease in the capability to eliminate impurities is considered inevitable. These synthetic ligands have not replaced protein A in industrial production, and are an inadequate solution to the problems of acidic elution buffer solutions.
(4) Methods Employing Protein A with an Acidic Elution Buffer Solution while Stabilizing the Antibody
As set forth above, it has not proved easy to develop an antibody purification method that can effectively replace protein A and acidic elution buffer solutions. Accordingly, methods of preventing the denaturation (structural change, association and aggregation reactions) of an antibody following contact with acidic elution buffer solution have been proposed.
Following the recovery of antibody bound to protein A with an acidic elution buffer solution, a stabilizing agent was immediately added in the form of a polyol compound such as polyethylene glycol, polyvinyl pyrrolidone, or ethylene glycol, to suppress association and aggregation reactions. See Japanese Patent Application No. Hei 2-273194. Based on this method, the addition of a stabilizing agent as soon as possible following contact with an acidic buffer solution suppresses the association and aggregation reactions, but the structural change at an acidic pH as set forth above cannot be suppressed. Furthermore, instead of using a stabilizer in the form of the polyol as proposed by Higuchi et al., a change in the pH itself must be prevented. See Carpenter et al., Pharmaceutical Research 14 (1997), 969-975. Accordingly, this technique is not a fundamental solution to the problems caused by acidic elution buffer solutions.
A method has been proposed wherein a buffer solution replacement column is serially connected to the rear section of a column packed with protein A-binding support so that the buffer solution of antibody desorbed from protein A is replaced with neutral pH buffer solution within as short a time as possible. See Japanese Patent Application Publication No. Hei 1-135798. Although this method minimizes contact time, the change in antibody structure itself is not prevented. Thus, in the same manner as the above-cited method of JPA No. Hei 2-273194, this technique does not constitute a fundamental solution to the problems caused by acidic elution buffer solutions.
Based on the above-described prior art, the issues involved in solving the problems caused by acidic elution buffer solutions can be set forth as follows.
Utilizing the high affinity of protein A for antibody Fc domains requires the use of protein A as the ligand binding the antibody. An antibody desorption enhancer must be added to adequately lower the affinity of protein A to antibody Fc in a neutral pH buffer solution, or be of an acidity mild enough to prevent denaturation of the antibody by association or aggregation following binding of the antibody.